The precise mechanisms through which astrocytes modulate synaptic transmission are not clear. It is also not clear how dysfunction in these cells, including those caused by drugs of abuse, can lead to brain disorders. The objective of this proposal is to develop a method that will allow the culture of astrocytes within a microfluidic platform to enable real-time measurements of intracellular Ca2+ concentration ([Ca2+]i) via fluorescent probes simultaneously with secretion of neuroactive substances in response to various stimuli. Guided by our strong preliminary data, we will obtain the objective of this proposal by pursuing the following specific aims: 1) monitoring D-serine and glutamate secretion simultaneous with [Ca2+]i imaging within astrocyte cultures, and 2) quantitation of ATP release from astrocyte cultures. In the first aim, we will develop a microfluidic system to monitor, with high time resolution, the release of primary amines from astrocytes simultaneous with intracellular [Ca2+]i imaging. In the second aim, we will integrate an enzymatic assay for adenosine triphosphate to allow monitoring of all the major gliotransmitters from astrocytes. The research proposed in this application is innovative because it will utilize a broad approach to the measurement of multiple factors released from astrocytes combined with simultaneous [Ca2+]i imaging, providing a highly dynamic view of gliotransmission. The results of this system will be significant because it will provide the first tool capable of investigating release of all major gliotransmitters simultaneously in an automated fashion in response to a number of stimulants. Ultimately, this device will produce a novel tool for future studies regarding astrocyte biology and the roles these cells have in neuroprotective behavior, tripartite synaptic transmission, and the effects of drugs of abuse.